Despite an improved molecular understanding of glioblastoma multiforme (GBM), there are no effective treatments to cure this brain cancer. Our overall goal is to develop oncolytic herpes viral (oHSV) strategies to kill glioblastoma stem cells (GSCs) and improve GBM therapy. This will likely require combinatorial therapy. In Aim 1 we extend our current studies of combinatorial systemic therapy and local oHSV anti-angiogenesis to GSCs and include a new anti-VEGF agent and two anti-angiogenic oHSV vectors. In Aim 2, we explore mechanisms involved in targeting GSCs in both the perivascular and hypoxic niches using novel small molecule inhibitors of hypoxia inducible factor (HIF) to increase treatment efficacy. In Aim 3, we utilize a syngeneic mouse GSC model to test whether the anti-angiogenic oHSV (G47?-mIL12) elicits an anti-tumor immune response in addition to lysing cancer cells and inhibiting tumor growth. We also hypothesize that anti- angiogenic therapy will enhance the immune response and anti-tumor efficacy of G47D-mIL12. The novelty of this proposal involves (1) the new human and mouse GSC models used; (2) the novel anti-angiogenic oncolytic herpes vectors (oHSV) available for study; (3) the synergistic mechanisms of tumor cell death involving both GSC and vasculature; (4) The mechanisms of oHSV killing of GSC in both the vascular niche and hypoxic niche of GBM; (5) the use of small molecule hypoxia inducible factor (HIF) inhibitors in conjunction with oHSV; (6) the ability of a vector to synergistically use multile avenues of tumor cell killing (oncolysis, anti-angiogenesis, inhibiting GSC to endothelial cell differentiation, initiating an anti-tumor immune response). The significance is that (1) therapeuti outcomes for GBM have advanced little in several decades; (2) we are defining mechanisms of synergy capable of killing the GSC population that has evaded most other therapies; (3) we have demonstrated our ability to produce vectors capable of going into clinical trial; (4) the strategies we are pursuing should be readily translatable to the clinic and we expect that these studies will lead to new clinical trials with increased efficacy for this fatal tumor. We started te concept of genetically engineered oncolytic viral (OV) therapy. For brain tumors, oHSV therapy has developed into several early phase clinical trials. For tumors outside the brain, OV therapy is now in Phase III clinical trials and is entering an era of increased interest by pharmaceutical companies for translation into mainstream cancer therapy. Herein, we explore important issues that have prevented GBM from being cured by standard therapies: How to kill the stem cell component; How to kill the hypoxic fraction of tumor; How to enhance killing of distant cells and provide lasting immunity. Our prior studies have led to several clinical trials and we expect the studies in this grant proposal to lead to improved approaches for these important problems and more effective treatment of people with GBM. PUBLIC HEALTH RELEVANCE: Genetically engineered oncolytic herpes viruses in combination with systemic pharmaceuticals are used to explore issues that have prevented glioblastoma from being cured by standard therapies: How to kill the stem cell component; How to kill the hypoxic fraction of tumor; How to kill distant cells and provide lasting immunity.